Tetramethylpyrazine was originally isolated from Ligusticum wallichii in 1957. Tetramethylpyrazine, whose tonality could be described as cocoa, coffee, lactose, vine, and galbanum, is wildly used as flavoring additive in food, tobacco, and alcohol industries. It is the main biologically active alkaloid existing in the Chinese herb Chuanxiong which improves blood circulation, tonifies qi and removes blood clots and is routinely used in China as medicaments for several diseases, such as heart-and-brain's vein disease, breath system disease and glomerular disease.
There are three methods, i.e. biosynthetic method, direct extraction from plants, and chemical synthetic method, for tetramethylpyrazine production.
As to biosynthesis of tetramethylpyrazine, the application of biotechnology mainly refers to microbial technology which includes screening and mutation of strains and fermentation technology. To date, there are some such reports out side of China and mainly in France, Japan, Germany, and the US.
Tetramethylpyrazine can be fermented from some kinds of sugars. The current research status of biosynthesis of tetramethylpyrazine is as follows.
Kosuge et al. firstly identified that some microbes are capable of pyrazine production and they found Bacillus subtilis was a tetramethylpyrazine producer in 1962. Now people know that some bacteria and epiphyte can synthesize different kinds of alkyl pyrazines (Seitz, 1994; Gallois, 1984). Yamaguchi et al. once tried to utilize these microorganisms in submerged fermentation but failed in industrialization because of the too low product concentration in fermentation broth (1993).
Itohiki-natto is one of the most popular fermentative foods due to its special pyrazine flavor. It is fermented from Bacillus natto on soybean solid medium by Japanese. However, the results were poor for solid-state fermentations were experiential methods. The yield of flavor substances was less than 22 μg/kg wet weight (Kosuge et al. 1971).
Besson et al. from France did a lot of work in this field. They yield 0.58 g/kg utilizing Bacillus subtilis IFO 3013 by solid-state fermentation on soybean medium (1998). This was the best result reported of tetramethylpyrazine biosynthetic methods. But there are still great efforts before the realization of industrialization.
Due to the low concentration of tetramethylpyrazine in plants, extraction of tetramethylpyrazine from plant materials is a costly technology and unfit for industrialization. While the method of chemical synthesis can bring in high yield of tetramethylpyrazine, it demands intensive reaction conditions and more sophisticated equipments. Moreover, the resulting tetramethylpyrazine is not regarded as natural product and there are serious problem in protecting environment. In contrast, the method of biosynthesis has the advantages such as natural product, low material costs, mild reaction conditions, environmentally benign and is considered as the future study of tetramethylpyrazine synthesis with very good prospects. However, isolation of tetramethylpyrazine producing strains from nature is a difficult job and yields from wild strains obtained by this way are usually low. As well as the low start base of other microbial ways, technologies of tetramethylpyrazine production by biosynthesis are immature and have not been industrialized.